Immunodeficiency, Centromeric instability and Facial anomalies (ICF) syndrome is an autosomal recessive disorder affecting multiple organ systems. Recent human genetic studies have identified that most ICF syndrome patients carry genetic mutations in DNMT3B encoding a de novo DNA methyltransferase. Consequently, cells derived from affected patients exhibited significantly reduced methyltransferase activity and abnormal hypomethylation of CpG sites within pericentromeric satellite, subtelomeric, and X chromosome regions. Transgenic mouse models that mirror genetic mutations in human ICF syndrome do not fully recapitulate the human pathogenesis of the syndrome. For this purpose, we propose to develop a human stem cell model that is better suited to understand the molecular basis of ICF syndrome. In Specific Aim 1, we will generate and characterize human iPSCs carrying ICF mutations and determine whether DNMT3B deficiency affects cell proliferation and genome stability. ICF-iPSCs will be characterized for expression of pluripotency markers and multi-potentials of cell differentiation through embryoid formation and teratoma formation. Specific Aim 2 is designed to compare directed differentiation of control and ICF mutant iPSCs into hematopoietic progenitor cells (HPCs) and neural crest lineage cells (NCs) to determine the impact of DNMT3B deficiency on lineage-specific cell differentiation. In Specific Aim 3, we will attempt to rescue ICF phenotypes by introducing wild-type DNMT3B expression in ICF mutant iPSCs, HPCs, and NCs. We will focus on understanding whether DNMT3B expression can rescue stage-specific DNA hypomethylation on the genome stability and gene expression during cell differentiation. Our proposed research will provide a novel approach to understanding the pathogenesis of ICF syndrome, thus potentially develop a new approach to cure ICF syndrome through stem cell therapy.